Continuous biochemical reactor for analysis of sub-picomole quantities of complex organic molecules

ABSTRACT

A novel reactor for reacting and subsequently analyzing sub-picomole quantities of a sample organic molecule. The reactor includes a continuous capillary connected between two valves that control fluid flow in the capillary. One part of the capillary forms a reaction chamber where the sample may be immobilized for subsequent reaction with reagents supplied through the valves. Another part of the capillary passes through or terminates in the detector portion of an analyzer such as an electrophoresis apparatus, liquid chromatographic apparatus or mass spectrometer. The apparatus may form a peptide or protein sequencer for carrying out the Edman degradation reaction and analyzing the reaction product produced by the reaction. The protein or peptide sequencer includes a reaction chamber for carrying out coupling and cleavage on a peptide or protein to produce derivatized amino acid residue, a conversion chamber for carrying out conversion and producing a converted amino acid residue and an analyzer for identifying the converted amino acid residue. The reaction chamber may be contained within one arm of a capillary and the conversion chamber is located in another arm of the capillary. An electrophoresis length of capillary is directly capillary coupled to the conversion chamber to allow electrophoresis separation of the converted amino acid residue as it leaves the conversion chamber. Identification of the converted amino acid residue takes place at one end of the electrophoresis length of the capillary.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application based on priorapplication Ser. No. 08/292,605, filed Aug. 18, 1994, and now U.S. Pat.No. 5,415,841, which was a continuation application of application Ser.No. 08/069,125, filed on May 28, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates to biochemical reactors and methods of operatingbiochemical reactors.

BACKGROUND AND SUMMARY OF THE INVENTION

Analysis of minute (sub-picomole) quantities of various organicmolecules, as for example proteins, oligosaccharides, peptides,nucleotides, amino acids and DNA is of great value in manyenvironmental, biotechnological, medical and pharmaceuticalapplications. In many cases, the available sample is very small,rendering analysis difficult and time consuming. Particularly this isthe case where a small quantity of protein or peptide has been isolatedand it is desired to identify the protein or peptide by determining thesequence of amino acids in the protein or peptide.

The basic chemistry of sequencing a peptide or a protein is known asEdman degradation chemistry, after P. Edman, Arch. Biochem. Biophys. 22,475 (1979). In the Edman degradation reaction, the first step is acoupling step in which a peptide or protein (hereinafter describedsimply as a peptide) is first treated with a peptide degradationcoupling agent such as phenylisothiocyanate (PITC), which couples to thepeptide or protein to form a coupled peptide. The next step is acleavage step in which the coupled peptide is treated with anhydrousacid, such as anhydrous trifluoroacetic acid (TFA), to cleave thecoupled peptide to produce an amino acid residue, such as cyclicthiazolinone amino acid (ATZ), and leaving a truncated peptide, thepeptide having had one amino acid residue cleaved from the peptide. Inthe next step, a conversion step, the amino acid residue is separatedfrom the truncated peptide and treated with an aqueous solution orconversion agent, typically an aqueous acid such as aqueous TFA, toproduce a converted amino acid residue, such as phenyl thiohydantoin(PTH) amino acid. The converted amino acid residue carries an amino acidthat has been cleaved from the peptide. In the final step,identification, the cleaved amino acid is identified by some appropriatemeans.

An example of an apparatus for carrying out the Edman degradationreaction and sequencing a protein or peptide is described in R. Hewicket al, A Gas-Liquid-Solid Peptide and Protein Sequencer, The Journal ofBiological Chemistry, Vol. 256, no. 15, Aug. 10, 1981, pp. 7990-7997. Inthe Hewick device as described in this paper, the sample peptide orprotein is immobilized in a reaction chamber having a diameter of about6 mm formed from a pair of facing conical cavities at the end of twofacing glass rods. Capillaries, having diameter of about 0.5 mm, in thecenters of the respective glass rods, supply reagent to and removeproducts from the reaction chamber. Coupling and cleavage are carriedout in the reaction chamber and the derivatized amino acid residue isremoved from the reaction chamber to a conversion flask, where theconversion step is carried out. The converted amino acid residue is thentaken from the conversion flask, and the converted amino acid isidentified by liquid chromatography. Further summary of the manner ofoperation of such an apparatus is described by M. W. Hunkapiller, inProtein/Peptide Sequence Analysis: Current Methodologies, A. S. Brown,ed., CRC Press Inc., Boca Raton La., 1988, at 87.

The entire degradation cycle of the Hewick apparatus requires in theorder of 45 minutes, and has limited sensitivity. Further, the Hewickdevice requires relatively large quantities of reagent, which reducesits effectiveness for analyzing very small (femtomole, or 10⁻¹⁵ mole, orless) quantities of converted amino acid residue, in effect rendering itincapable of sequencing less than 1 picomole (10⁻¹² mole) of peptide.

The inventors have proposed a novel reactor for reacting andsubsequently analyzing sub-picomole quantities of a sample organicmolecule. The reactor includes a continuous capillary connected betweentwo valves that control fluid flow in the capillary. One part of thecapillary forms a reaction chamber where the sample may be immobilizedfor subsequent reaction with reagents supplied through the valves.Another part of the capillary passes through or terminates in thedetector portion of an analyzer such as an electrophoresis apparatus,liquid chromatographic apparatus or mass spectrometer.

The apparatus may form a peptide or protein sequencer for carrying outthe Edman degradation reaction and analyzing the reaction productproduced by the reaction. The protein or peptide sequencer includes areaction chamber for carrying out coupling and cleavage on a peptide orprotein to produce derivatized amino acid residue, a conversion chamberfor carrying out conversion and producing a converted amino acid residueand means for identifying the converted amino acid residue. In oneaspect of the invention, unlike in the Hewick device, the reactionchamber is contained within one arm of a capillary and the conversionchamber is located in another arm of the capillary. In a further aspectof the invention, an electrophoresis length of capillary is directlycapillary coupled to the conversion chamber to allow electrophoresisseparation of the converted amino acid residue as it leaves theconversion chamber. Identification of the converted amino acid residuetakes place at one end of the electrophoresis length of the capillary.

In one aspect of a method according to the invention, the Edmandegradation reaction is carried out in a capillary. Immobilization,cleavage and coupling of the peptide/protein takes place in a reactionchamber portion of the capillary to produce derivatized amino acidresidue, and conversion takes place in a conversion chamber portion ofthe capillary to produce a converted amino acid residue. Electrophoresisseparation of the converted amino acid residue then preferentially takesplace in an electrophoretic length of the same capillary, including byapplying an electric field across the conversion chamber, followed byidentification of the converted amino acid residue.

BRIEF DESCRIPTION OF THE DRAWINGS

There will now be described preferred embodiments of the invention, withreference to the drawings, by way of illustration, in which likenumerals denote like elements and in which:

FIG. 1 is a schematic of one embodiment of a biochemical reactor andanalyzer according to the invention, showing the reactor enlarged;

FIG. 2 is a schematic of another embodiment of a biochemical reactor andanalyzer according to the invention;

FIG. 3 is a schematic of another embodiment of a biochemical reactor andanalyzer according to the invention;

FIG. 4 is a section through a reaction chamber for use with thebiochemical reactors shown in FIGS. 1, 2, 3, 6, 9 and 10;

FIG. 5 is a section through another reaction chamber for use with thebiochemical reactors shown in FIGS. 1, 2, 3, 6, 9 and 10;

FIG. 6 is a schematic of another embodiment of a biochemical reactor andanalyzer according to the invention having no conversion chamber (secondreaction chamber);

FIG. 7 is a schematic of an analyzer for use with any of the reactorsshown in FIGS. 1, 2, 3, 6, 9 or 10;

FIG. 8 is a graph showing results of use of the analyzer in FIG. 7;

FIG. 9 is a schematic of an embodiment of a biochemical reactor andanalyzer according to the invention in which the analyzer is a massspectrometer;

FIG. 10 is a schematic of another embodiment of a biochemical reactorand analyzer according to the invention in which the analyzer is a massspectrometer;

FIG. 11 is a schematic showing a portion of an embodiment of theinvention for use in association with a mass spectrometer electrospraydevice as the analyzer; and

FIG. 12 is a schematic showing an embodiment of the invention in whichthe analyzer uses liquid chromatography for analysis.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used in this patent document, a capillary is a small tube or pipedefined by one or more members of arbitrary cross-sectional shapesuitable for fluid flow, such as circular. A capillary for use inanalyzing sub-picomole quantities of sample should be no more than 1 mminside diameter (circular bore) and preferably less than 530 μm insidediameter. A sample is the material that is to be analyzed or sequenced,and in the case of sequencing a peptide or protein is the peptide orprotein to be sequenced. After reaction of the sample with a reagent,the product of the reaction will be referred to as a reaction product,which in the case of peptide or protein sequencing is the amino acidresidue.

Referring to FIG. 1 there is shown a schematic of a combined reactor andanalyzer specifically designed for peptide and protein sequencing. Thereactor is shown enlarged at 10 and the analyzer is shown at 12. Thereactor 10 and analyzer 12 are connected by an arm of a primarycapillary 14 that provides a continuous flow path between the reactor 10and analyzer 12. The analyzer 12 is located at one end 16 of the primarycapillary 14 and is used for identification of a sample. A pneumaticallyactuated multi-position distribution valve 22, such as are used forcapillary liquid chromatography and as may be obtained for example fromValco Instruments Co. Inc. of Houston, Tex., is connected at its commoninlet/outlet port to a reaction end 18 of the primary capillary 14 andis used for delivery of reagents into the primary capillary 14. Otherports of the valve 22 are connected via suitable lines 24 such as Teflontubes to vials 26 (containing fluids identified by the identifiers R1,R2, R3, R4, S1, S2 and S3, where S refers to solvent and R refers toreagent), to a source of argon gas Ar, to waste through line 55 or to avacuum pump 53, according to the requirements of the reaction to becarried out in the reactor. As known in the art, a suitable inert gas isused to pressurize the vials 26 and deliver the reagent into thecapillary 14. The delivery of small volumes of reagent may be managedusing the split injection technique of Novotny (V. L. McGuffin and M. V.Novotny, Anal. Chem., 55, 580 (1983)). The valve 22, lines 24 and vials26 together with the waste line 55, vacuum pump 53 and argon source forma means for controlling fluid flow in the capillary 14. Except asotherwise stated in this document, each valve located at the end of acapillary is a multi-position distribution valve of the same type asused for valve 22, as for example valve 17 connected to theidentification end 16 of the capillary 14. The arm of the capillary 14connecting the reaction arm 28 to the identification end 16 ispreferably a fused silica tube having 50 μm inside diameter (ID) and 190μm outside diameter (OD).

A glass fibre reaction mat 32, or other suitable means for holding asample such as a peptide or protein in the capillary 14, is fixed withinarm 28 of the primary capillary 14 adjacent the reaction end 18. Thelocation of the glass fibre reaction mat 32 defines a reaction chamber20 within the capillary. A polymeric quaternary ammonium salt, such asPolybrene™ (hexadimethrine bromide), is impregnated into the reactionmat 32. The Polybrene attaches to the reaction mat and to peptides orproteins (or other samples) to immobilize the peptides or proteins inthe reaction chamber 20 while allowing reagents to flow through thereaction mat 32. The reaction mat 32 may be held in place in thecapillary 14 as shown in FIGS. 4 or 5 for example. Other chemicalmethods of attachment of a peptide may also be used, as for example forsolid phase sequencing, methods as described in Aebersold, R., CovalentAttachment of Peptides for High Sensitivity Solid Phase SequenceAnalysis, Analytical Biochemistry, 187, pp. 55-65, 1990, may be used.

In FIG. 4, the reaction mat 32 is sandwiched between two pieces of fusedsilica capillary 28 (for example, 100 μm ID, 245 μm OD) over which ispushed a short piece of fused silica capillary 34 having for example 250μm ID. The pushing of the sandwiched mat into the capillary havinglarger inside diameter may be used to effectively cut the mat. A heater36 formed for example of a thermocouple 38 (Peltier device), with brassheat sink 40, connected by leads 42 to a suitable power source 44, islocated about the capillary arm 28 at the reaction chamber 20.

In FIG. 5, the capillary 28 is formed from a 50 μm ID, 360 μm ODcapillary 28a on one side of the reaction chamber 20 and a 400 μm ID,525 μm OD, fused silica capillary 28b on the other side, held togetherwith epoxy 35. Glass fibre filter disc 32 pre-cycled with polybrene, isplaced on top of a circular porous PTFE membrane 33, both resting andheld by force of gravity on the top of the capillary 28a and bycompression against the sides of the capillary.

In cases where derivatives of a reacted sample are directed immediatelyto the identification end of the capillary for analysis (as for examplewhere identification of only the N-terminal end of a peptide isrequired), the capillary 14 may require no other inlet for reagentsother than valve 22 (see FIG. 6). However, in the case where for examplea protein or peptide is to be sequenced using the Edman degradationreaction, and the manner of analysis of the amino acid requires use of afluid (electrophoresis medium for example) that will react with thepeptide or protein, then it is necessary to isolate the peptide orprotein in the reaction chamber during analysis, while the fluid may beflushed through the rest of the capillary 14.

In such a situation, a supply arm 48 of the capillary 14 having one endmeeting the reaction arm 28 at junction 50 may be used for delivery ofreagent. Isolation of the reaction arm 28 of the capillary 14 may beobtained by a suitable valve on the arm 28 between the reaction chamber20 and the junction 50 or simply by closing of the valve 22. Closing ofthe valve 22 will immobilize fluids in the reaction arm 28 of thecapillary due to the forces binding the fluid to the capillary wall. Avalve 52 connected via lines 24 to vials 26, similar to the valve 22, issituated at the other end of the supply arm 48. The valve 52 may also besupplied with a vacuum pump 53 for use in evacuating the supplycapillary during peptide or protein sequencing and waste 55 for drainingfluids from the capillary 14. The supply arm 48 and the identificationend 16 of the primary capillary 14 form a continuous capillary. Reagentsor solvents delivered through valve 22 may be flushed to waste eitherthrough valve 52 or valve 17. Reagents and buffer solutions, used forexample in electrophoresis, delivered through valve 52 may be flushedthrough valve 17 to waste.

Junction 50 may be made in several ways. It may use a commerciallyavailable T for attaching several fused silica capillaries together, inwhich T the capillary arms 28, 48 and 16 are inserted, the capillaryarms all therefore comprising the continuous capillary 14. Or thecapillary arms 28, 48 and 16 may be made of a unitary capillary with itsarms fused together at the junction 50. Alternatively, the capillary 14and the junction 50 can be formed by etching the capillary into a faceof a solid block of inert material such as glass that abuts against aface of another block of inert material. Such a capillary can be formedby conventional micromachining techniques.

For electrophoresis separation of reaction product in the primarycapillary 14, it may be desirable to convert reaction product into aform suitable for electrophoresis. In such a case, the supply arm 48(FIG. 1), or the primary capillary 14 between the identification end 16and the junction 50 (FIG. 2), may be provided with means to hold thereaction product in the supply capillary 14 for conversion. Such a meansmay be a thermocouple cooler 60 (Peltier device) made in accordance withthe design shown for the heater 36 in FIG. 4, but with the polarity ofthe leads reversed to provide for cooling rather than heating. Thecooler 60 may be used to freeze reaction product in the supply arm 48,where it may be subject to further reaction or conversion. The locationof the thermocouple 60 therefore defines a conversion chamber 62 in thesupply capillary 48 or in the primary capillary 14.

In FIG. 2, which shows the conversion chamber in the primary capillary14 between the junction 50 and the identification end 16, valve 66 islocated at the identification end and valve 68 is located on the supplycapillary 48. A vacuum pump 53 is attached to valve 66 for removingvapour from the conversion chamber 62 and valve 66 is also supplied withlines 24 and vials 26 for delivery of reagents and solvents to thesupply capillary 48, while valve 68 is likewise supplied with lines 24and vial 26.

For use in electrophoresis separation of the sample, a high voltagepower supply 64 is connected to the valves 22 and 52 (FIG. 1), valves 22and 66 (FIG. 2) or valves 22 and 70 (FIG. 3) at which electrodes (notshown) are applied to the electrophoresis medium in known manner. Whenthe supply capillary 48 is not being used then the power supply may beconnected to solution in the primary capillary via valve 22 as shown inFIG. 3. The length of capillary being used for electrophoresis must ofcourse have electrophoretic length, that is, a length over whichsufficient separation of the reaction product takes place so that it isidentifiable.

A further embodiment of a peptide or protein sequencer is shown in FIG.3. In this case, the supply capillary 48 connects as in FIG. 1 and 2 tothe primary capillary 14 to form the junction 50, and also connects tothe primary capillary 14 at reaction arm 28 between the reaction chamber20 and valve 22 (same as the valve 22 shown in FIG. 1 but additionalinlet lines 24 have been hooked up to the valve). A valve 70 is used forflushing and applying a vacuum to the identification end 16 of theprimary capillary. A switching valve 72 at the junction of the capillary48 and the reaction arm 28 permits fluids from valve 22 to be directedalong either arm 28 or arm 48. Valve 22 therefore may be used to providethe same reagents as valve 52 in FIG. 1. By this means, onemulti-position distribution valve may be omitted from the sequencer andreplaced with a simple switching valve.

A more generalized reactor and analyzer are shown in FIG. 6. A capillary14 extends continuously between a valve 22 and a valve 70, both of themulti-position distribution type described above as suitable for use asvalve 22 in FIG. 1, with lines 24 and vials 26 respectively forproviding reagent and solvents to the capillary 14, together with linesfor application of a vacuum through pump 53 to the capillary 14, as wellas for allowing waste to be removed from the capillary 14. The capillary14 has a reaction end 18 and an identification end 16. The reaction end18 includes a heater 36, such as is described in FIG. 4, and a holdingmeans for holding a sample such as the reaction mats described in FIGS.4 and 5. The holding means is located in a portion of the capillary 14which thus forms a reaction chamber 20. An analyzer 12 formed forexample of a laser 76 or other suitable light source and detector 78 isshown at the identification end, and the capillary 14 passes through thedetection zone of the analyzer.

In general, following electrophoresis separation of a sample in theprimary capillary 14, a laser 76 may be used to illuminate the sampleand allow detection of the sample using a detector 78 using conventionalelectrophoresis methods such as those described in Jorgenson et al,Capillary Zone Electrophoresis, Science vol. 222, 266-272, 1983; Chenget al, Subattomole Amino Acid Analysis by Capillary Zone Electrophoresisand Laser Induced Fluorescence, Science, vol. 242, pp. 562-564, 1988; Wuet al, High Sensitivity Fluorescence Detector for FluoresceinIsothiocyanate Derivatives of Amino Acids Separated by Capillary ZoneElectrophoresis, Journal of Chromatography, 494, 1989, 141-155; Rohliceket al, Simple Apparatus for Capillary Zone Electrophoresis and itsApplication to Protein Analysis, Journal of Chromatography, 494, 1989,87-89; Yu et al, Atttomole Amino Acid Determination by Capillary ZoneElectrophoresis with Thermooptical Absorbence Detection, Anal. Chem.,61, 1989, 37-40; Sweedler et al, Fluorescence Detection in CapillaryZone Electrophoresis Using a Charged Coupled Device with Time DelayedIntegration, Anal. chem. 63, 1991, 496-502; Monnig et al, On-columnSample Gating for High Speed Capillary Zone Electrophoresis, Anal.Chem., 63, 1991, 802-807; Deyl et al, Design of a Variable Wavelength UVAbsorption Detector for On-column Detection in Capillary Electrophoresisand Comparison of its Performance to a Fixed Wavelength UV AbsorptionDetector, Journal of Liquid Chromatography, 12(13), 1989, 2527-2561;Swerdlow et al, Capillary Gel Electrophoresis for DNA Sequencing,Journal of Chromatography, 516, 1990, 61-67; Waldron et al, CapillaryZone Electrophoresis Separation and Laser Based Detection of BothFluorescein Thiohydantoin and Dimethylaminoazobenzene ThiohydantoinDerivatives of Amino Acids, Electrophoresis, 11, 1990, 777-780; Bruno etal, Thermooptical Absorption Detection in 25 μmid Capillaries: CapillaryElectrophoresis of Cansl-Amno Acids Mixtures, Applied Spectroscopy, vol.45, no. 3, 1990, 462-367; Swerdlow et al, Three DNA Sequencing MethodsUsing Capillary Gel Electrophoresis and Laser Induced Fluorescence,Anal. Chem., 63, 1991, 2835-2841; and Wu et al, Capillary ZoneElectrophoresis Separation and Laser-Induced Fluorescence Detection ofZeptomole Quantities of Fluorescein Thiohydantoin Derivatives of AminoAcids, Talanta, vol. 39, no. 2, 173-178, 1992. The detector 78 and laser76 together form an analyzer. Various analyzers may be used inconjunction with the reactor and analyzer combination described here.

A sample analyzer 12 or identification means is shown in FIG. 7. Aportion of the identification end 16 of capillary 14 is shown incross-section. The capillary 14 (50 μm ID, 190 μm OD) is illuminatedwith a laser 80 that is selected for absorption of its output by thesample carried by electrophoretic medium within the capillary 14, suchas a 5 mW average power 10-μJ pulse energy KrF excimer waveguide laser(Potomac Photonics Model GX-500) operating at λ=248 nm, 610 Hz pulserepetition rate and 50 ns pulse width. The laser beam is focused with a15 mm focal length quartz biconvex lens at right angles to thecapillary, the location of the laser beam thus defining a detection zoneof the analyzer through which the capillary passes. If the capillary 14has a polyimide coating, it should be removed as for example by burningwith a gentle flame. The output beam of a second laser 82, such as a 3mW helium-neon laser (Melles Griot Model 05-LHP-151), is directed atright angles to both the capillary 14 and the beam from the laser 80,and focused with a 7x lens. For convenience, the beam may be reflectedone or more times with mirrors, such as mirror 83. A transducer 84, forexample a 1 mm² silicon photodiode, is located in the beam path of thelaser 82 at the other side of the capillary 14 from the laser 82, about30 cm from the capillary. An electric signal from the transducer 84 isconditioned with a current to voltage converter (1 M ohm feedbackresistor in parallel with a 47 pF capacitor) and supplied to anamplifier 86. The exemplary amplifier is a two-phase lock-in amplifier(Ithaco Model 3961) phase referenced to the excimer laser pulserepetition rate. Data from the lock-in amplifier 86 is supplied to aprocessor 88 (for example a personal computer). The data may beprocessed by any of several known methods, as for example using Matlabsoftware to convolute the data with a gaussian filter.

Sample amino acids in the buffer solution in the capillary 14 areselectively excited by the radiation from the excimer laser andrelaxation from the excited state heats the buffer solution, resultingin a change in the index of refraction of the solution and theconsequent bending of the laser beam from the helium neon laser 82. Thedeflection of the laser beam is recorded as a change in intensity oflight detected by transducer 84.

The data is in the form of electromagnetic signals. The magnitude of thesignals depends on the amount of light detected by the transducer, whichin turn depends on the index of refraction of the electrophoreticmedium, which depends on the degree to which it is heated, which in turndepends on the amount of amino acid with light absorptioncharacteristics in the electrophoretic medium. Hence the data isrepresentative of the amount of amino acid in the electrophoretic mediumat a particular time.

Results of using the apparatus shown in FIG. 7 for the detection ofPTH-amino acids are shown in FIG. 8. In this case, the length ofcapillary 14 before the detector was 34 cm. A voltage of 8 kV was placedacross the capillary. A 12.5 mM pH 7.0 borate/phosphate buffercontaining 35 mM sodium dodecyl sulfate was used for electrophoresismedium. Electrokinetic injection of 5 s at 500 V was used. Stocksolutions of 10⁻² concentration of the amino acids alanine (A), arginine(R), asparagine (N), aspartic acid (D), cysteic acid (C), serine (S),glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine(I), leucine (L), PTH-PTC lysine (K), methionine (M), phenylalanine (F),proline (P), threonine (T), tyrosine (Y), tryptophan (W) and valine (V)were prepared by dissolving each amino acid in a 50% acetonitrile and50% mM phosphate/borate buffer. Mixtures of amino acids were prepared bypipetting 10 μL aliquots into 1.5 mL of 12.5 mM pH 7.0 borate/phosphatebuffer that contained 35 mM sodium dodecyl sulfate. The peaks shown inFIG. 8 are identified according to the letter in parenthesis listedafter each amino acid noted above. A 1.1 mV baseline signal is due toabsorbance of the excimer laser beam by trace impurities in theseparation buffer. The disturbance at 4.5 min. is due to the elution oftrace amounts of acetonitrile, added to the analyte to effectdissolution. The arrival time of a specific peak from the analysis of anunknown amino acid may be compared with the arrival times of known aminoacids shown in the graph of FIG. 8 and thus identify the amino acid.

The electrophoretic separation was by micellar electrophoresis, asdescribed in Otsuka et al, Electrokinetic Chromatography with MicellarSolutions Separation of Phenylthiohydantoinp-Amino Acids, 2. Chromatogr.332, 1985, p. 219-226. Micellar electrophoresis is particularly usefulwhen the amino acid residue is not charged. The buffer fluid have movesin one direction in the electric field established by the high voltagesource 64, while micelles in the buffer fluid have electrophoreticmobility in an opposite direction. The amino acid residue is partitionedin and out of the micelles, and during partitioning is carried with theflow of buffer. Each amino acid has a different partition coefficientwhich governs the length of time it remains in the micelle. Hence, thelength of time the amino acid takes to travel along the capillary is anindication of the type of amino acid. The results shown in FIG. 8 werenot obtained using the apparatus of FIGS. 1, 2 or 3 but are believedrepresentative of results that could be obtained by the repetitivesequencing of a peptide or protein using the apparatus of any of FIG. 1,2 or 3.

Other analyzers may be used. When FITC is used as the degradationcoupling agent in the Edman reaction, the analyzer may use capillaryzone electrophoresis and direct identification of FTC (fluoresceinthiocarbamyl) following well known techniques as for example describedby Wu, S. et al, Capillary Zone Electrophoresis Separation and LaserInduced Fluorescence Detection of Zeptomole Quantities of FluoresceinThiohydantoin Derivatives of Amino Acids, Talanta, Vol. 39, No. 2, pp.173-178, February 1992. The fluorescence detector may use a sheath flowcuvette design as described in Cheng et al, Subattomole Amino AcidAnalysis by Capillary Zone Electrophoresis and Laser InducedFluorescence, Science, vo. 242, pp. 562-564, October 1988.

The analyzer 12 may also use high performance liquid chromatography, orother types of liquid chromatography, such as described in Kettler etal, Pulsed-Laser Photothermal Refraction Detection in Capillary LiquidChromatography, Anal. Chem. 1987, 59, 1733-1736, or mass spectrometry.

If a detector is used which requires the consumption of the reactionproduct by the analyzer, such as in electrophoresis using a sheath flowcuvette or a mass spectrometer, then the reaction product and thesolution carrying it must be taken out of the capillary 14 into theanalyzer.

An example of such a device is shown in FIGS. 9 and 10. In FIG. 9, valve92, with inlet lines 24 and vials 26, has its common outlet connected toa reaction arm 28 of a capillary 14. A thermocouple 36 is placed about aportion of the arm 28 and defines a reaction chamber 20 in the capillaryarm 28. Means to immobilize a sample to be reacted is located in thereaction chamber 20 for example as shown in FIGS. 4 or 5. A supplycapillary arm 48 leads from junction 51 to valve 94, with lines 24 andvials 26. On the supply capillary 48 is a thermocouple 60 defining aconversion chamber 62 in the supply capillary. At an identification end96 of the capillary 14, a valve 98 is located, with the end 96terminating in the ionization chamber of a mass spectrometer 102. Themass spectrometer may be a triple quadrupole mass spectrometer sold bySciex Division of MDS Health Group Limited, of Thornhill, Ontario,Canada, under its trademark TAGA 60000E. Ionization of reaction productfrom the reaction chamber may be enhanced using the techniques describedin U.S. Pat. No. 4,935,624 to Henion et al. The overall structure andoperation of the apparatus shown in FIG. 9 is similar to the operationof the apparatus shown in FIG. 1, except that a line 100 for carryingmass spectrometer buffer liquid or gas from a vial 26 is provided on thevalve 98 that can be added to the reaction product in capillary 14 fordelivery into the mass spectrometer 102 for analysis.

FIG. 10 shows a device similar to the device of FIG. 6, used for examplefor amino acid analysis and N-terminal amino acid identification. As inFIG. 6, there is no means to isolate the reaction chamber duringelectrophoresis with the consequence that application of the buffer willhydrolize any truncated peptide or protein and prevent further analysis,apart from a first amino acid that has been cleaved from the protein orpeptide. Capillary 14 extends between valve 104 and valve 106, each withlines 24 and vials 26 and has a thermocouple 36 (or other heater) at aportion of the capillary defining a reaction chamber 20. A high voltagepower supply 64 is placed across the valves 104 and 106 with electrodesconnectable to buffer solution supplied through the line at vialsindicated by S3. Vacuum is provided from pump 53 through line 55 andwaste through line 57. Sheath buffer or gas for mass spectrometry isprovided through line 100. The capillary 14 continues through the valve106 (through internal capillary lines within the valve) to massspectrometer 102, similar to the mass spectrometer shown in FIG. 9.

If an electrospray device is used for the delivery of reaction productto the mass analyzer, then a slightly different structure must be used.In FIG. 11 there is shown a modification of the design shown in FIG. 9.The design is the same except that an electrospray device 108 is shownon the mass spectrometer, such as is described in Henion et al, U.S.Pat. No. 4,935,624. The tip 110 of the capillary 14 extends into themass spectrometer ionization chamber 112. The tip 110 of the capillary14 will in this case be conducting, and typically the capillary in thisportion will be made of stainless steel. A lead 114 from the highvoltage source 64 connects to the tip 110 of the capillary 14 thusestablishing an electric potential through the electrophoretic medium tovalve 94. Separation of amino acids takes place in the capillary 14,while analysis takes place in the mass spectometer 102.

FIG. 12 shows a further embodiment of the invention in which liquidchromatography is used for analysis. Valve 22 has its common portconnected to a reactor end 18 of capillary 14. The valve 22 has variousof its inlet ports connected to lines 24 leading to vials 26 containingreagents (R) or solvents (S). One inlet port is connected to a line 120leading to a gradient mixer 122, which is supplied aqueous buffer (A)and an organic modifier (B2) from vials 130 through lines 128 and pumps126. Since liquid chromatography does not use electrophoresis, no highvoltage source is required. If isochratic chromatography is used, onlyone pump is required. The reaction end 18 of the capillary 14 includes athermocouple 36 and reaction chamber 20, like the reaction chamber 20shown in FIG. 4 or 5. The capillary 14 passes through the detection zoneof an analyzer formed by laser or equivalent light source 76 anddetector 78. At least the portion 134 of the capillary 14 between thereaction chamber 20 and the detection zone of the analyzer is filledwith liquid chromatographic packing material, such as coated silicabeads. The capillary should have an inner diameter suitable forreceiving the packing material, such as 250 μm. The other end 16 of thecapillary 14 terminates in a valve 132, having inlet ports connected vialine 24 to vial 26 and to a vacuum pump 53 and waste 55. FIG. 12 showsthe set up for liquid chromatography when used for amino acid analysisor other analysis in which hydrolysis of the sample after a firstreaction step is not a concern. If a peptide is sequentially reacted inthe reaction chamber, as for example in Edman degradation, then a supplyor bypass capillary such as the capillary shown in FIGS. 1, 2, 3 or 9would be required with suitable modifications to the valves. Inaddition, rather than a light source 76 and detector 78, a massspectrometer as shown in FIG. 10 could be attached to an inlet port ofvalve 132.

The manner of operation of the various apparatus referred to in FIGS. 1,2 or 3 is now described. It will be appreciated that the basic chemistryis known in the art so that the details of the chemistry will not bedescribed. In the case of each of the apparatus shown, the vials 26 havebeen labelled according to their use in amino acid identification,including peptide or protein sequencing in the case of the apparatusdescribed in FIGS. 1, 2 and 3. The following table indicates the use ofthe vials:

    ______________________________________                                        Label     Use                                                                 ______________________________________                                        R1        Coupling agent, for example PITC                                    R2        TMA (trimethylamine)                                                S1        Wash fluid, eg Ethyl acetate                                        S2        Solvent, for example benzene                                        S3        Electrophoresis medium (buffer soln)                                B1        Mass spectrometer buffer                                            R3        Anhydrous acid, for example TFA                                     R4        Aqueous acid, for example TFA                                       Ar        Source of inert gas, for example argon                              Vacuum    Indicates a pump for evacuating the capillary.                      Waste     A drain for removing fluids from the capillary                      ______________________________________                                    

Referring to FIGS. 1, 2 or 3, firstly, the sample peptide or protein isloaded onto the reaction mat 32 and the reaction chamber heated to about60° C. Using valve 22 for input and valves 52, 66 and 70 for waste,peptide degradation coupling agent, for example PITC, is then introducedinto the reaction arm 28 with heptane and the reaction mat dried underargon. In the case of the apparatus shown in FIG. 3, the switch valve 72is switched for the duration of coupling and cleavage to isolate thesupply capillary 48 from the primary capillary 14 during coupling andcleavage. TMA is then introduced to reaction arm 28 to promote formationof coupled peptide. Unwanted material may be flushed out with ethylacetate input via valve 22 to waste through valves 52 (FIG. 1), 66 (FIG.2) or 70 (FIG. 3). Anhydrous acid (TFA), either as a pulsed liquid orsaturated vapour in argon, is introduced to reaction arm 28 and thereaction chamber 20 where it cleaves the amino acid residue from thecoupled peptide to produce amino acid residue and leaving a peptide thathas been truncated by one amino acid. Throughout these steps, valve 17in FIG. 1 is held closed. A like process is used for the apparatus ofFIGS. 2 and 3, although in the case of FIG. 3, valve 17 is not present.

Once the amino acid residue (reaction product) has been produced, it isextracted from the reaction mat 32 using a solvent such as benzene, witha freezing point around 0° C., introduced via valve 22 through thereaction arm 28. The benzene, with some of the amino acid residuedissolved in it, is transported into the supply capillary 48 or theidentification arm 16 in the case of the apparatus of FIGS. 2 or 3 whereit is frozen into place using the thermocouple 60. A vacuum imposed onthe supply capillary 48 through one of valve 52 (FIG. 1), 66 (FIG. 2) or70 (FIG. 3) may then be used to sublime the solvent, leaving the aminoacid residue frozen into the conversion chamber 62. The conversionchamber may then be raised to room temperature (by reversing thepolarity of the power applied to the thermocouple 60) and a small slugof aqueous acid is introduced through valve 52 to valve 17 (FIG. 1),valve 68 to valve 53 (FIG. 2) or valve 22 to valve 70 (FIG. 3) toconvert the ATZ amino acid residue to PTH amino acid residue (secondreaction product) in the conversion chamber. During conversion, thereaction chamber 20 must be isolated to avoid hydrolysis of thetruncated peptide, such as by closing valve 22 (and closing off thereaction chamber using valve 72 in the apparatus of FIG. 3). Theconversion chamber 62 is again frozen using the thermocouple 60 and theaqueous acid removed by vacuum through one of valves 52, 66 or 70. Againthe conversion chamber is brought to room temperature using thethermocouple 60.

The next step is identification or analysis. The supply capillary 48 andidentification arm 16 of the capillary 14 are filled with aqueous pH 7buffer or other capillary electrophoresis buffer from valve 22 throughvalve 17 (FIG. 1), valve 68 to valve 66 (FIG. 2) or valve 22 to valve 70(FIG. 3, after switching of the valve 72 to isolate the reaction chamber20). Valve 52, valve 68 or valve 22 is then switched to a separationbuffer containing acetonitrile, sodium dodecyl sulfate and 20 mM pH 7aqueous buffer. Potential is then applied across the conversion chamberfrom valve 52 through valve 17 (FIG. 1), valve 68 to 66 (FIG. 2) orvalve 22 to valve 70 with one valve held at an electrophoretic potentialsuch as ±8 kV to induce electrophoresis separation of amino acids in thecapillary with the other valve grounded. The distance between theconversion chamber and the analyzer 12 should be an electrophoreticlength in which sufficient separation of the amino acid residue willtake place to allow analysis. The amino acid is identified using one ofvarious analyzers at the identification end 16.

A program for the apparatus shown in FIG. 1 is set out at the end ofthis disclosure in Tables 2, 3, 4 and 5. Table 2 shows the function ofeach of the valve positions for the valves of FIG. 1 (Valve Acorresponds to valve 22, valve B to valve 52 and valve C to valve 17).Table 3 shows the time sequence of steps in the program. Peltier1 isheater 36. Peltier2 is thermocouple 60. Spellman HV is the high voltagesource 64. CZE means capillary zone electrophoresis. Table 4 shows theinside and outside diameters and the length of the capillary tubinghooked up to the various valve positions or if Teflon tubing is usedinstead of capillary tubes. The valves may be automatically programmedfor these steps if desired using hardware provided by the manufacturer.For the apparatus of FIG. 6, a like process is followed. However, in thecase of amino acid analysis or N-terminal amino acid analysis or similarsuch analyses, where isolation of the peptide or protein or other sampleis not required, the conversion step is not required and anelectrophoretic potential can be applied across the reaction chamberafter formation of the reaction product and filling of the capillarywith an appropriate buffer. Separation then occurs as with conventionalelectrophoresis, followed by analysis at the analyzer. In the case ofthe apparatus shown in FIGS. 9, 10, 11 and 12, like steps of the Edmandegradation reaction may be carried out. The method steps for theapparatus of FIGS. 9 and 11 are analogous to those of FIG. 2, and themethod steps of FIGS. 10 and 12 are analogous to those of FIG. 6, withthe exception that mass spectrometry buffer must be added to thesolution through line 100 following electrophoresis for use of theapparatus of FIG. 10 and liquid chromatographic identification using theaqueous buffer A and organic modifier B2 in conventional manner must becarried out for use of the apparatus of FIG. 12 following degradation.That is, for liquid chromatography, after amino acid residue has beenproduced by the Edman degradation reaction, the chamber is dried,leaving only amino acid residue in the reaction chamber. Then aqueousbuffer A and organic modifier B2 is pumped through the gradient mixer122 into capillary 14, with a gradually increasing ratio of modifier tobuffer. Amino acids attach to the liquid chromatographic packingmaterial in the capillary 14 and as the modifier to buffer ratio isincreased, amino acids selectively detach from the packing material andpass through the detection zone of the analyzer whence they can beidentified in known manner.

The following is a comparison of an embodiment of the present inventionwith a commercial sequencer following the Hewick apparatus design:

                  TABLE 1                                                         ______________________________________                                                        COMMERCIAL   PRESENT                                          ITEM            SEQUENCER    SEQUENCER                                        ______________________________________                                        reaction chamber volume                                                                       150 μL    0.2 μL                                        glass-fibre filter area                                                                       450 mm.sup.2 0.5 mm.sup.2                                     max. sample volume                                                                            30 μL     0.04 μL                                       gas reagent flow rate                                                                         3 mL/min.    0.05 mL/min                                      liquid reagent flow rate                                                                      0.5 mL/min.  abt                                                                           0.004 mL/min.*                                   cycle duration  45-60 min    20-30 min                                        minimum amount sequence-                                                                      1 picomole   1 femtomole                                      able                                                                          ______________________________________                                         *(flow rates differ slightly depending on viscosity of solvent and tubing     I.D.)                                                                    

The reaction chamber volume defined in Table 2 is the volume in whichthe reaction mat sits.

A person skilled in the art could make immaterial modifications to theinvention described and claimed in this patent without departing fromthe essence of the invention. Thermocouples need not be used for heating(thermocouple 36) nor for heating and cooling (thermocouple 60), ratherother heating and cooling techniques could be used. For holding thereaction product in the conversion chamber, a packed bed ofchromatographic material, such as silica beads, could be fixed in thecapillary using such techniques as sintering. A like technique could beused for immobilizing the sample in the reaction chamber, with Polybreneapplied to the bed of beads. The apparatus is not limited to use foranalysis of samples in solution. The apparatus may also be used forsolid phase sequencing.

While multi-position valves have been described as being used for thevalves 22, 53 etc., it is possible to use miniature syringe pumps forthe fluid flow control means, and various types of multi-position valvesmay be used. The capillary 14 may be a silica tube made of one or moremembers or etched in a glass block for example. For the carrying out ofsome reactions, for example the Edman degradation reaction, a valve withrandomly accessible ports may be preferred, rather than a valve in whichthe valves must be used sequentially. For carrying out the Edmandegradation reaction using the degradation agentsdimethylaminoazobenzene isothiocyanate (DABITC) or fluoresceinisothiocyanate (FITC), or with combinations of PITC with FITC or DABITC,additional ports are required beyond the ports described in FIG. 1 forexample to handle the DABITC or FITC. If necessary, an additional valvemay be used, also connected to the reaction end of capillary 14. Theprogram outlined in Table 3 would be used, modified for the particulardegradation agent used.

                  TABLE 2                                                         ______________________________________                                        Valve A positions                                                                        Valve B positions                                                                             Valve C positions                                  ______________________________________                                        1.  12% TMA    1.    waste       1.  plug                                     2.  PITC       2.    vacuum      2.  waste                                    3.  Ar         3.    plug        3.  plug                                     4.  12% TMA    4.    Ar          4.  waste                                    5.  Ar         5.    25% TFA     5.  CZE buffer                                                                    (14 psi)                                 6.  Ethyl Acetate                                                                            6.    Ar          6.  CZE buffer +                                                                  HV                                       7.  Ar         7.    plug        7.  waste                                    8.  TFA vapour 8.    vacuum      8.  vacuum                                   9.  Ar         9.    plug        9.  waste                                    10. Benzene    10.   CZE buffer (3.5 psi)                                                                      10. plug                                     11. Ar         11.   CZE buffer (no Ar)                                                                        11. plug                                     12. plug       12.   Ar (14 psi) 12. plug                                     ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Valve A   Valve B   Valve C   Notes                                           ______________________________________                                        --        --        --        Peltier1 on, heat                                                             (65 C.) 1.40 v                                  1. 12% TMA                                                                              1. waste  1. plug   Peltier1 (0:40 min)                             2. PITC   "         "         Peltier1 (0:03 min)                             3. Ar     "         "         Peltier1 (2:00 min)                             4. 12% TMA                                                                              "         "         Peltier1 (7:30 min)                             5. Ar     "         "         Peltier1 (2:00 min)                                       "         "         Peltier1 off                                    6. Ethyl  "         "         (0:06 min)                                      Acetate                                                                       7. Ar     "         "         (3:00 min)                                      "         "         "         Peltier1 on, heat                                                             (48 C.) 0.94 v                                  8. TFA vapour                                                                           "         "         Peltier1 (6:00 min)                             "         "         "         Peltier1 off                                    9. Ar     "         "         (2:00 min)                                      "         "         "         Peltier2 on, freeze                                                           (-10 C.) 1.45 v                                 10. Benzene                                                                             "         "         Peltier2 (0:10 min)                             11. Ar (75/                                                                             "         "         Peltier2 (1:00 min to                           360 um)                       push benzene)                                   12. plug  "         "         Peltier2 (1:00 min to                                                         push benzene)                                             2. vacuum "         Peltier2 (3:00 min to                                                         remove benzene)                                 "         3. plug   "         Peltier2 (3:00 min to                                                         remove benzene)                                 "         4. Ar     "         Peltier2 (3:00 min to                                                         remove benzene)                                 "         "         2. waste  Peltier2 (1:00 min to                                                         dry)                                            "         5. 25%    "         Peltier2 (0:05 min)                                       TFA                                                                 "         6. Ar (75/                                                                              "         Peltier2 (0:08 min to                                     360 um)             push TFA thru)                                  "         7. plug   "         Peltier2 (0:08 min to                                                         push TFA thru)                                  "         "         3. plug   Peltier2 (0:08 min to                                                         push TFA thru)                                  "         "         "         Peltier2, heat (65 C.,                                                        10:00 min)                                      "         "         "         Peltier2 on, freeze                                                           (-10 C.)                                        "         8. vacuum "         Peltier2 (4:00 min to                                                         remove aq TFA)                                  "         9. plug   "         Peltier2 (4:00 min to                                                         remove aq TFA)                                  "         10. CZE   "         Peltier2 (0:05 05 min, fill                               buffer              to Peltier 2)                                   "         10. CZE   4. waste                                                            buffer                                                              "         10. CZE   5. CZE    Peltier2 (2:25 min, fill                                  buffer    buffer (14                                                                              to Peltier 2)                                                       psi)                                                                10. CZE   5. CZE    Pettier 2 off                                             buffer    buffer (14                                                                    psi)                                                      "         10. CZE   6. CZE                                                              buffer    ber +                                                                         HV                                                        "         11. CZE   6. CZE                                                              buffer, no                                                                              buffer +                                                                      HV                                                        "         11. CZE   6. CZE    Spellman HV on, start                                     buffer, no                                                                              buffer +  CZE prog.                                                           HV                                                        "         12. plug  6. CZE                                                                        buffer +                                                                      HV                                                        "         "         7. vacuum (4:00 min to remove                                                           buffer)                                         "         "         8. Ar (14                                                                     psi)                                                      "         1. waste  8. Ar (14 (2:00 min to dry)                                                   psi)                                                      "         "         9. plug                                                   "         "         10. plug                                                  "         "         11. plug                                                  "         "         12. plug                                                  "         "         1. plug   2nd cycle starts                                ______________________________________                                    

                                      TABLE 4                                     __________________________________________________________________________    Valve A positions Valve B positions  Valve C positions                        __________________________________________________________________________      12% TMA                                                                               75/360 × 60 cm                                                                  1.                                                                              waste    98/230 × 30 cm                                                                  1.                                                                              plug                                     PITC   250/340 × 55 cm                                                                  2.                                                                              vacuum  teflon tubing                                                                          2.                                                                              waste    75/360 × 25 cm            Ar      75/360 × 60 cm                                                                  3.                                                                              plug             3.                                                                              plug                                     12\% TMA                                                                              75/360 × 60 cm                                                                  4.                                                                              Ar      teflon tubing                                                                          4.                                                                              waste    75/360 × 25 cm          5.       250/340 × 58 cm                                                                  5.                                                                              25% TFA 250/340 × 40 cm                                                                  5.                                                                              CZE buffer (14                                                                        250/340 × 30 cm                                                 psi)                                     Ethyl Acetate                                                                        250/340 × 50 cm                                                                  6.                                                                              Ar       75/360 × 40 cm                                                                  6.                                                                              CZE buffer +                                                                          250/340 × 25 cm                                                 HV electrode                             Ar      75/360 × 50 cm                                                                  7.                                                                              plug             7.                                                                              waste   250/340 × 20 cm            TFA vapour                                                                            75/360 × 53 cm                                                                  8.                                                                              vacuum  teflon tubing                                                                          8.                                                                              vacuum  teflon tubing                    Ar     250/340 × 50 cm                                                                  9.                                                                              plug             9.                                                                              waste   250/340 × 20 cm          10.                                                                             Benzene                                                                              250/340 × 47 cm                                                                  10.                                                                             CZE buffer (3.5                                                                       250/340 × 40 cm                                                                  10.                                                                             plug                                                       psi)                                                        Ar      75/360 × 50 cm                                                                  11.                                                                             CZE buffer (no                                                                        250/340 × 40 cm                                                                  11.                                                                             plug                                                       Ar)                                                         plug            12.                                                                             Ar (14 psi)                                                                           teflon tubing                                                                          12.                                                                             plug                                   __________________________________________________________________________

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A combined reactor withanalyzer for reacting and analyzing a sample, the reactor and analyzercomprising:(a) a continuous capillary including:(i) a primary capillaryportion having a reaction end and an identification end, first fluidflow control means being disposed at the reaction end and second fluidflow control means being disposed at the identification end; and (ii) asupply capillary portion having a first end and a second end, the firstend being connected to the primary capillary portion between thereaction end and the identification end to form a junction, and thesecond end having third fluid flow control means for supplying fluid tothe supply capillary portion and to the identification end of theprimary capillary portion; (b) sample holding means at a first selectedportion of the reaction end of the primary capillary portion forselectively holding a sample within the reaction end of the primarycapillary portion for reaction to form a reaction product, the firstselected portion defining a reaction chamber; and (c) identificationmeans at the identification end for identifying the reaction product. 2.The combined reactor and analyzer of claim 1 further including:reactionproduct holding means at a second selected portion of the supplycapillary portion or the primary capillary portion between the thirdfluid flow control means and the identification end for holding thereaction product for further reaction.
 3. The combined reactor andanalyzer of claim 2 in which:the reaction product holding means islocated at the supply capillary portion between the junction and thethird fluid flow control means.
 4. The combined reactor and analyzer ofclaim 2 in which the identification means includes:a laser forirradiating the reaction product in the primary capillary portion; meansfor detecting fluorescence from irradiated reaction product; and meansfor establishing an electric field in electrophoretic medium in thesupply capillary portion and primary capillary portion along anelectrophoretic length of the supply capillary portion and the primarycapillary portion while isolating the reaction chamber from theelectrophoretic medium.
 5. The combined reactor and analyzer of claim 2in which the identification means includes:means for electrophoreticallyseparating the reaction product by passage through an electrophoreticmedium; a first laser for exciting the reacted sample in a portion ofthe electrophoretic medium with laser light that is selectively absorbedby the reaction product; a second laser for directing a beam of light atthe reacted sample; and means for detecting deflection of the beam oflight due to heating of the electrophoretic medium following excitationof the reaction product.
 6. The combined reactor and analyzer of claim 2further including a valve across the primary capillary portion betweenthe reaction chamber and the junction between the supply capillaryportion and the primary capillary portion, whereby isolation of thereaction chamber from the identification end of the primary capillaryportion may be ensured.
 7. The combined reactor and analyzer of claim 2in which the reaction product holding means is formed from a coolingdevice disposed about the supply capillary portion.
 8. The combinedreactor and analyzer of claim 2 in which:the reaction product holdingmeans is located at the primary capillary portion between the junctionand the second fluid flow control means.
 9. The combined reactor andanalyzer of claim 8 in which:the second end of the supply capillaryportion is connected to the primary capillary portion at the reactionend; and the third fluid flow control means includes a valve fordirecting fluids from the first fluid flow control means to one of theprimary capillary portion and the supply capillary portion.
 10. Thecombined reactor and analyzer of claim 2 in which the reaction productholding means is formed from a cooling device disposed about one of thesupply capillary portion and the primary capillary portion.
 11. Thecombined reactor and analyzer of claim 2 in which the primary capillaryportion has an inside diameter of less than 530 μm.
 12. The combinedreactor and analyzer of claim 3 in which the identification meansincludes:a laser for irradiating the reaction product in the primarycapillary portion; means for detecting fluorescence from irradiatedreaction product; and means for establishing an electric field inelectrophoretic medium in the supply capillary portion and primarycapillary portion along an electrophoretic length of the supplycapillary portion and the primary capillary portion while isolating thereaction chamber from the electrophoretic medium.
 13. The combinedreactor and analyzer of claim 3 in which the identification meansincludes:means for electrophoretically separating the reaction productby passage through an electrophoretic medium; a first laser for excitingthe reacted sample in a portion of the electrophoretic medium with laserlight that is selectively absorbed by the reaction product; a secondlaser for directing a beam of light at the reacted sample; and means fordetecting deflection of the beam of light due to heating of theelectrophoretic medium following excitation of the reaction product. 14.The combined reactor and analyzer of claim 3 further including a valveacross the primary capillary portion between the reaction chamber andthe junction between the supply capillary portion and the primarycapillary portion, whereby isolation of the reaction chamber from theidentification end of the primary capillary portion may be ensured. 15.The combined reactor and analyzer of claim 3 in which the reactionproduct holding means is formed from a cooling device disposed about thesupply capillary portion.
 16. The combined reactor and analyzer of claim8 in which the identification means includes:a laser for irradiating thereaction product in the primary capillary portion; means for detectingfluorescence from irradiated reaction product; and means forestablishing an electric field in electrophoretic medium in the supplycapillary portion and primary capillary portion along an electrophoreticlength of the supply capillary portion and the primary capillary portionwhile isolating the reaction chamber from the electrophoretic medium.17. The combined reactor and analyzer of claim 8 in which theidentification means includes:means for electrophoretically separatingthe reaction product by passage through an electrophoretic medium; afirst laser for exciting the reacted sample in a portion of theelectrophoretic medium with laser light that is selectively absorbed bythe reaction product; a second laser for directing a beam of light atthe reacted sample; and means for detecting deflection of the beam oflight due to heating of the electrophoretic medium following excitationof the reaction product.
 18. The combined reactor and analyzer of claim8 further including a valve across the primary capillary portion betweenthe reaction chamber and the junction between the supply capillaryportion and the primary capillary portion, whereby isolation of thereaction chamber from the identification end of the primary capillaryportion may be ensured.
 19. The combined reactor and analyzer of claim 8in which the reaction product holding means is formed from a coolingdevice disposed about the supply capillary portion.
 20. The combinedreactor and analyzer of claim 1 in which the identification means is amass spectrometer having an ionization chamber and the identificationend of the primary capillary portion of the continuous capillaryterminates in the ionization chamber of the mass spectrometer.
 21. Thecombined reactor and analyzer of claim 2 in which the identificationmeans is a mass spectrometer having an ionization chamber and theidentification end of the primary capillary portion of the continuouscapillary terminates in the ionization chamber of the mass spectrometer.22. The combined reactor and analyzer of claim 3 in which theidentification means is a mass spectrometer having an ionization chamberand the identification end of the primary capillary portion of thecontinuous capillary terminates in the ionization chamber of the massspectrometer.
 23. The combined reactor and analyzer of claim 8 in whichthe identification means is a mass spectrometer having an ionizationchamber and the identification end of the primary capillary portion ofthe continuous capillary terminates in the ionization chamber of themass spectrometer.
 24. The combined reactor and analyzer of claim 1 inwhich the identification end of the primary capillary portion includesliquid chromatographic packing material.
 25. The combined reactor andanalyzer of claim 2 in which the identification end of the primarycapillary portion includes liquid chromatographic packing material. 26.The combined reactor and analyzer of claim 3 in which the identificationend of the primary capillary portion includes liquid chromatographicpacking material.
 27. The combined reactor and analyzer of claim 8 inwhich the identification end of the primary capillary portion includesliquid chromatographic packing material.
 28. The combined reactor andanalyzer of claim 20 in which the identification end of the primarycapillary portion includes liquid chromatographic packing material. 29.The combined reactor and analyzer of claim 21 in which theidentification end of the primary capillary portion includes liquidchromatographic packing material.
 30. The combined reactor and analyzerof claim 22 in which the identification end of the primary capillaryportion includes liquid chromatographic packing material.
 31. Thecombined reactor and analyzer of claim 23 in which the identificationend of the primary capillary portion includes liquid chromatographicpacking material.